Witnessing of conspecifics in pain has been
shown to elicit socially triggered freezing in
rodents. It is unknown how robust this response
is to repeated exposure to a cage-mate
experiencing painful stimulation. To address
this question, shock-experienced Observer rats
repeatedly witnessed familiar Demonstrators
receive painful footshocks (six sessions).
Results confirm that Observers freeze during the
first testing session. The occurrence of this
behaviour however gradually diminished as the
experimental sessions progressed, reaching
minimal freezing levels by the end of the
experiments. In contrast, the appearance and
continuous increase in the frequency of
yawning, a behavior that was inhibited by
metyrapone (i.e,. a glucocorticoid synthesis
blocker), might represent an alternative coping
strategy, suggesting that the observer's reduced
freezing does not necessarily indicate a
disappearance in the affective response to the
Demonstrator's distress.

Empathy, the ability to understand and share
the feelings of others, can be conceptualized as
a hierarchically organized multi-level capacity
such that higher-level processes are built on
top of more primal ones, like emotional
contagion [1;2]. Emotional contagion is
an automatic tendency to converge with another
individual's emotional state as a direct
consequence of perception and without
distinguishing the origin of the emotion (i.e.,
self vs other) [3;4]. An increasing
amount of evidence suggests that at least the
basic components of empathy, namely emotional
contagion, are shared with non-human
mammals.

Evidence of emotional state sharing in
rodents originates from studies showing
socially-induced hyperalgesia [5],
social priming [6] and social buffering
[7,8]. For example, mice and rats learn
to fear a conditioned stimulus by simply
observing or interacting with a conspecific in
distress [6]. Sharing the distress of
others has also been evidenced by the socially
triggered freezing response exhibited by
shock-experienced observers when witnessing
demonstrators endure painful foot electroshocks
[9&endash;13]. This phenomenon is
modulated by the genetic characteristics of the
rodent strain [14], context [12]
the degree of familiarity between observer and
demonstrator and by previous experience of the
observers [11]. In addition, the
expression of emotional contagion in both mice
and humans is dependent on stress levels of the
observer animal [15], as reduced
affective responses can be restored by
administration of a glucocorticoid synthesis
inhibitor.

So far, all the studies investigating
emotional contagion in rodents have examined the
behavior of animals after a single exposure or
interaction with another in distress. Despite
the increasing characterization of emotional
contagion in rodents, it is still unknown how
animals would respond following repeated
witnessing of a conspecific in pain. How would
animals respond following repeated testing with
a conspecific in pain? This question is of
methodological interest, as many experiments
would require repeated testing of an individual,
be it to investigate, using repeated measures
the effect of drugs or to record neural activity
and behavior over multiple exposures. Here,
leveraging our rodent model of empathy
[9], we exposed shock experienced rats
on multiple days to familiar Demonstrators
undergoing footshocks to investigate whether and
how behavior changed from day to day.

Results and Discussion

Shock-experienced Observer rats were exposed
to familiar Demonstrators undergoing footshocks
during six identical, semi-consecutive testing
days. Each testing day contained a preshock
baseline period and 5 shocks, during which the
freezing of Demonstrators and Observers was
scored.

Freezing of Demonstrators

A 6 Days x 6 Epochs (1 baseline + 5 shock
periods) ANOVA revealed a significant Day x
Epoch interaction (F25,100 = 3.96, p<0.0001).
This interaction was driven by Day 1, as
removing Day 1 rendered the interaction
non-significant (p>0.05). A 6 Day repeated
measures ANOVA on baselines showed differences
between freezing levels of Demonstrators during
the baseline period of Days 1 to 6 (F5,30 =
10.751, p<0.001; Fig 1B). Specifically,
relative to the preshock-baseline of Day 1,
Demonstrators displayed high freezing levels
throughout (paired t-tests of baseline Day 1
compared to Days 2 to 6, all p<0.05).
Further, planned comparisons using paired sample
t-tests of each shock period to baseline,
revealed that on Day 1, Demonstrators froze more
following each one of the five shocks than
baseline (all p<0.01; Fig 1B). In contrast,
the same analysis showed that except for two
data points, on Days 2 to 6, the Demonstrators'
freezing levels during the shock periods was not
higher than on baseline. This indicated that
after the first test Day Demonstrators developed
contextual freezing. Freezing of Observers

A 6 Days x 6 Epochs ANOVA on the freezing of
Observers revealed main effects of Days (F5,20 =
9.45, p<0.0001) and Epoch (F5,20 = 4.1,
p<0.01; Fig 1B) and a significant Day x Epoch
interaction (F5,20 = 1.68, p<0.05). In
contrast to Demonstrators, a 6 Day repeated
measures ANOVA on baselines showed no
significant differences between freezing levels
during the baseline period of Days 1 to 6
(p>0.1). Planned paired sample t-tests
comparing shock with baseline periods for each
Day indicates diminished freezing after Day 4
(Fig 1B). Specifically, post-hoc t-tests
revealed Observers froze significantly more
during various shock- periods of Days 1, 2 and 3
(compared to those day's baseline, all
p<0.05) while this was not the case on Days 4
to 6 (all p>0.4). This reduction in freezing
of Observers, could suggest that their
sensitivity to the distress of the Demonstrator
diminishes following repeated exposure.
Emergence of Yawning in Observers

Throughout the experiment, we however
noticed the appearance of yawning, an
unusual behaviour displayed only by the
Observers (Fig 2, S1 Video). Yawning
included extensive opening of the mouth and most
of the times a full-body extension and upward
pointing of the snout. A 6 day repeated measures
Friedman test on normalized yawns (i.e., number
of yawns during the first 10mins of shock period
minus number of yawns during preshock period
[first 10 minutes at start of test prior to
1st shock]) detected differences in
yawning frequency between Days
(É'2 (5) = 12.12, p = 0.033). Planned
Wilcoxon posthoc tests comparing yawning
frequency between Day 1 and Days 2 to 6 revealed
that Observers yawned more during Day 6 compared
to Day 1 (p = 0.017). The yawning
response of Observers appeared to be specific to
the distress of the Demonstrators, as a Wilcoxon
signed rank test comparing the total number of
yawns during the combined preshock periods of
Days 1 to 6 to the total number of yawns during
the shock periods of Days 1 to 6, showed that
Observers yawned more during the shock periods
(Z(6) = -2.2, p<0.05) (Fig 1B and 1C).
Further, the percent of Observers yawning
increased throughout the experiment, with more
than 70% of animals yawning in the last
two Days compared with 0% on Day 1 (Fig 1C).
Moreover, by Day 6, all Observers had yawned at
some point indicating a generalized behavioral
response. The number of yawns depended on the
time of day at which rats were tested. Three
rats were tested in the morning (beginning of
their dark phase), and showed on average a total
of 9 yawns over the 6 days. Four rats were
testing in the afternoon (second half of their
dark phase) and showed on average a total of 19
yawns. To ensure that the increase of yawns
across Days was not due to an inadvertent shift
in testing time, the total number of yawns on
each Day was compared with the average testing
time on that day, but the relationship was
clearly non-significant (r2<0.0067, p =
0.87). Importantly, the results of a separate
experiment showing that yawning was
significantly inhibited by pre-treatment with
metyrapone (t(18) = 2.191, p = 0.042), suggests
that yawning reflects heightened stress
levels in the Observers.

Attention in Observers

To investigate whether the attention of
Observers towards the Demonstrators changed
throughout the experiment, the percent of time
Observers spent in the window zone (i.e., 12cm x
25cm area closest to the divider from the
Demonstrator' chamber) was quantified (Fig 1C).
A repeated measures ANOVA with 6 Days x 2 Epochs
(comparing the 10 minute preshock period prior
to shock start and the 10 to 15 minute after the
1st shock) revealed a main effect of Epoch (F1,4
= 13.3, p<0.05). This indicated that
Observers were drawn to spend a higher percent
of time in the window zone following shock
delivery. The absence of an interaction of Epoch
and Day (p>0.05) however shows this effect to
be constant across days, suggesting that the
attention of Observers was captured by the
Demonstrators receiving shocks in a way that was
sustained throughout the experiment.

Discussion

Demonstrators submitted to repeated and
unavoidable foot shocks exhibit elevated
freezing levels. Confirming previous findings
[9;11;12;16] in the first testing
session (i.e., Day 1), shock-experienced
Observers display freezing in response to
familiar Demonstrators enduring painful
footshocks. As to our core question of whether
these effects can be measured over repeated
days, as necessary for designs that require
repeated testing, our results show that freezing
gradually diminishes as a consequence of
repeatedly witnessing the Demonstrator receive
painful stimulations. Results of a separate
pilot experiment revealed that this reduction of
freezing occurs even if a number of experimental
factors are manipulated to reduce such
habituation (S1 Fig). Alternating between
different testing contexts, increasing the
length of time between testing sessions, pairing
two Demonstrators to each Observer and adding a
reminder shock session for the Observers, failed
to avoid a progressive reduction of the
Observer's freezing. From an experimental design
point of view, our findings thus indicate that
socially triggered freezing, as an assay of
social sensitivity in rodents may be more suited
for between-subject designs (that do not require
multiple testing of a given rodent) than for
within-subject designs. In addition, at first
glance, our findings could suggest that the
affective response of Observers to the distress
of Demonstrators is progressively reduced
throughout the testing sessions. The appearance
of yawning however complicates this
interpretation.

Yawning is a phylogenetically old
behavior, ubiquitously present across
vertebrates [17&endash;21]. It is
characterized by an extensive and involuntary
opening of the mouth with deep, prolonged
inspirations and short expirations lasting
approximately 10 seconds and commonly
accompanied by stretching. Yawning has
been observed in stressful situations in
different species like monkeys
[22&endash;25], rats [26] and
birds [27]. Thus, the emergence of
yawning as freezing becomes infrequent
could mean that animals change the way they
manifest their affective response to the
distress of others, but that animals are still
responsive to the distress of the other.
Yawning as a possible indicator of
elevated stress levels in the Observers was
confirmed by the reduction of this behavior
following administration of an anti-stress drug
(i.e., metyrapone) on test days 5 and 6. This is
in agreement, with results showing that
administration of anxiogenic drugs to monkeys
induces both anxiety-like behavior and
yawning [25], suggesting that
indeed yawning is indicative of elevated
stress levels. It has been hypothesized that in
addition to being a marker of arousal,
yawning marks the transition between
different types of arousal or levels of arousal
[28]. Perhaps then, yawning in
Observers indicates a change in the type of
arousal the Observers experience during the
first testing days compared to the arousal in
the last testing days. In contrast, the repeated
daily shock exposure the Demonstrators undergo
maintains a highly elevated but similar arousal
type throughout the experiment, thus preventing
yawning form emerging. However, this is
highly speculative and further testing such as
corticosterone measurements throughout the
experiment would be necessary to confirm this
hypothesis. In addition to the postulated role
in stress, yawning has been linked to a
variety of other functions, such as a
thermoregulation [20,29]. Here though
all sessions were conducted in conditions of
constant room temperature. That yawning
appeared following repeated testing, was
specific to the shock periods and only present
in Observers suggests that the most parsimonious
explanation for its emergence is that it is
indicative of changes in autonomic regulation
and reflects an affective response in the
Observer to the distress of the Demonstrator.
Together, these findings and the results showing
that the Observers attention towards the
Demonstrators is unaltered indicate that
although the freezing response to witnessed
distress changes over time, the affective
response of the Observers may outlast socially
triggered freezing and invite yawning as
a coping mechanism. However, more data will be
needed to fully understand the relationship
between freezing, yawning and other
stress related manifestations.